Protein ubiquitination is a highly conserved post-translational modification that regulates fundamental cellular processes.1-3 Ubiquitin conjugation is controlled by the sequential action of three enzymes: ubiquitin activating enzyme 1 (E1, 2 known), ubiquitin conjugating enzyme 2 (E2, ˜37 known), and ubiquitin ligase enzyme 3 (E3, ˜600 known).3 Among these, E3 enzymes stand out due to the astonishing complexity and diversity of biochemical reactions they catalyze. E3 enzymes control polyubiquitin chain linkages and polyubiquitin chain length, select specific substrates and specific residues to be ubiquitinated, as well as select and activate specific E2˜Ub thioesters for subsequent ubiquitin transfer events.4 Such complexity makes it difficult to study the biochemical properties of E3 enzymes, and to design assays to discover and to characterize pharmacological modulators of E3s. Typical biochemical assays to study E3 enzymes require at least three enzymes E1/E2/E3, ubiquitin, and ATP in the simplest case. The situation is more complex in the case of multi-subunit E3s such as cullin-RING E3s and APC/C E3, where up to 3-15 protein subunits are required to assemble the functional E3 enzyme.5-7 As such, studying protein ubiquitination and developing therapeutics targeting protein ubiquitination are difficult due to the complexity of the E1→E2→E3 ubiquitination cascade.